Myosin-binding protein C regulates the sarcomere lattice and stabilizes the OFF states of myosin heads

Muscle contraction is produced via the interaction of myofilaments and is regulated so that muscle performance matches demand. Myosin-binding protein C (MyBP-C) is a long and flexible protein that is tightly bound to the thick filament at its C-terminal end (MyBP-CC8C10), but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7) to myosin heads and/or the thin filament. MyBP-C is thought to control muscle contraction via the regulation of myosin motors, as mutations lead to debilitating disease. We use a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. We show that cleavage leads to alterations in crossbridge kinetics and passive structural signatures of myofilaments that are indicative of a shift of myosin heads towards the ON state, highlighting the importance of MyBP-CC1C7 to myofilament force production and regulation.


Supplementary Figure
Supplementary Figure 1.SNOOPC2 mouse line design and evaluation.a SNOOPC2 mice express a modified fMyBP-C that contains a TEV protease recognition site (red rectangle) and a SnoopTag (cyan trapezoid).The addition of TEV protease cleaves the endogenous C1-C7 domains of fMyBP-C while SnoopTag-C8-C10 remains anchored to the thick filament.Although not employed in this study, it is possible to incubate with a recombinant fMyBP-C construct containing the SnoopCatcher tag (green trapezoid), which will lead to in situ replacement of the cleaved fMyBP-C with the recombinant fragment.b Western blot of slow MyBP-C paralog from homozygous and wildtype SNOOPC2 psoas, before and after TEV protease treatment.As expected, no cleavage was detected in the slow MyBP-C paralog.This experiment was repeated with similar results three times, two are shown.c Representative image of a muscle fiber attachment for a mechanics experiment.The muscle fiber was glued between a motor and force transducer using 100% pure silicone rubber.d Passive tension-sarcomere length before (-) and after (+) TEVp treatment, with passive tension normalized to maximal tension at SL 3.0.e Tension-pCa relationship at SL 2.4, 2.7, and 3.0 before (-) and after (+) TEVp treatment, with every condition scaled to its maximum (pCa 4.5) tension.f Absolute tension-pCa relationship at SL 2.4, 2.7, and 3.0 before (-) and after (+) TEVp treatment.Statistics throughout are repeated-measures ANOVA designs followed by a Tukey Honestly Significant Difference (HSD) post-hoc test on significant main effects.Statistical differences are reported via connecting letters, where conditions assigned to different letters are significantly different.Data throughout reported as mean ± SE.The dataset was generated from n=49 fibers prepared from N=10 mice (6 female / 4 male).Further statistical details are in Supplementary Tables 2, 3, 5-8.Supplementary Figure 2. Control dataset for small-angle X-ray diffraction experiments.a-i X-ray diffraction patterns were collected from wildtype SNOOPC2 psoas fibers under passive conditions at two difference sarcomere lengths (SL), before (blue) and after (red) TEV protease treatment.X-ray features are shown for d10 (a), I11/I10 (b), σd (c), SM3 (d), SM6 (e), ST3 (f), SA6 (g), SA7 (h), and SgActin (i).As expected, we detected no changes in structural features in wildtype muscles caused by TEV protease treatment.Statistics throughout are ANOVA designs with main effects treatment, SL, and their interaction, and a random effect of individual, followed by a Tukey Honestly Significant Difference (HSD) post-hoc test on significant main effects (P < 0.05), and reported in figures as connecting letters: conditions assigned different letters are significantly different.Data throughout reported as mean ± SE.Dataset generated from n=20 fiber bundles prepared from N=6 mice (3 male / 3 female).Full descriptive and statistical details are in Supplementary Table 7. Supplementary Figure 3. Exemplar 1D intensity profiles of diffraction patterns.Representative 1D intensity profiles from the meridional (a-c) and equatorial (d-f) axes, for each sarcomere length (SL), before (solid lines) and after (dashed lines) TEV protease cleavage.Supplementary Table 1.Statistical details from experiments shown in Fig. 1g-i.N = number of preparations included in the condition.For SL 2.4, a total of 10 trials were run from psoas collected from 4 mice (2 female / 2 male).For SL 2.7, a total of 10 (9 post) trials were run from psoas collected from 6 mice (4 female / 2 male).For SL 3.0, a total of 10 trial were run from psoas collected from 4 mice (3 female / 1 male).

Supplementary Tables
Parameter  1g-h and Supplementary Fig. 1e.N = number of preparations included in the condition.For SL 2.4, a total of 10 trials were run from psoas collected from 4 mice (2 female / 2 male).For SL 2.7, a total of 10 (9 post) trials were run from psoas collected from 6 mice (4 female / 2 male).  1 k-m, and Supplementary Figure 1e.N = number of preparations included in the condition.For SL 2.4, a total of 10 trials were run from psoas collected from 4 mice (2 female, 2 male).For SL 2.7, a total of 10 (9 post) trials were run from psoas collected from 6 animals (4 female, 2 male).For SL 3.0, a total of 10 trials were run from psoas collected from 4 animals (3 female/ 1 male).

Parameter
Parameter

Table 2 .
Statistical details from experiments shown in Fig.

Table 10 . Statistical details from experiments shown in Figure 3. N = number of preparations included in the condition. A total of 37 trials were run from psoas collected from 15 mice (9 male /6 female).
Bold values indicate significant effect.SE = standard error of the mean